The goal of this project is to develop and evaluate methods for manufacturing dendritic cells (DCs) for clinical immunotherapy trials. In FY 1999, we developed and optimized a full-scale GMP method for 5-day flask culture of autologous DCs in RPMI, autologous plasma or allogeneic serum, IL4 and GMCSF, starting with peripheral blood monocytes collected by apheresis and purified by elutriation. The immature DCs generated are then available for further manipulations (e.g., peptide pulsing) prior to clinical administration. This manufacturing method has been successfully used in three clinical trials, two for pediatric sarcoma and one for colon cancer. A manuscript describing this method has been accepted by Cytotherapy. Because of our interest in developing closed systems and eliminating reagents that are difficult to standardize, we evaluated a 7-day culture system in a protein-defined, serum-free medium (XVIVO15) starting with monocytes from elutriation vs. negative immunomagnetic selection using the Isolex 300I, in bags vs. flasks. We demonstrated that the 2 different isolation methods for monocytes product equivalent immature DC populations, and that bags were equivalent to flasks. Furthermore, historical comparison showed that serum-free medium was equivalent and perhaps even superior to serum-containing medium for generation of immature DCs. A manuscript describing this work is in preparation. Ongoing development studies are now evaluating culture conditions for generating mature DCs using CD40 ligand after culture in IL4 and GMCSF; this will result in a process that will be put into clinical trial by early FY 2001. This project has required simultaneous development and evaluation of assays for quantitation and characterization of these cell populations. Extensive flow cytometric phenotyping of these cultured cells has led to the development of a flow cytometric panels that will continue to be evaluated in the context of clinical trials.